1. Technical Field
The present invention is related to obtaining a functionally intact whole gene directly from a genome, said gene being suitable for expression of gene products. The present invention further relates to a method of excising a complete gene from a genome in a single step and cloning the gene thus obtained.
2. Discussion of the Prior Art
Applicants are the first to develop a novel method of obtaining a complete functionally intact gene in a single step, as disclosed herein infra. Hence, there is no prior art known to the Applicants which is directly comparable to the present invention. However, current protocols for cloning genes utilizes one of three methods to prepare DNA for cloning: (1) The preparation of complementary DNA (cDNA) from messenger RNA (i.e., copying the mRNA into DNA); (2) shearing of genomic DNA into randomly broken fragments; and (3) cutting of genomic DNA into reproducible fragments with restriction endonucleases.
Once DNA fragments have been prepared, they are ligated into viral or plasmid vectors where they are reproduced. If the DNA contains a gene which codes for a protein product (most often this is the commercial reason to clone the DNA in the first place), the expression (production) of that protein or some part of it by the clone of interest is used to detect the gene-containing clone. A commonly used method to detect these protein products is binding of antibodies specific for the protein product.
The cDNA technique, which is most commonly used for preparing DNA for cloning, has the following limitations: (1) In almost every case only a part of the gene is cloned; (2) The piece of the cloned gene is usually biased toward the part which codes for the carboxyl terminal end of the protein; (3) The technique depends on a ready supply of mRNA which is only made, in most cases, in certain tissues or life cycle stages which may be difficult to obtain.
The present invention obviates the problems of the cDNA technique by cloning the complete gene in a single step. This reduces the time needed to get the complete gene, which of course, can also be obtained after cDNA cloning, but by going on to the restriction endonuclease method of preparing DNA for cloning and repeating the cloning process. Since the method of detection of a clone may be biased away from the structures displayed in the protein at the carboxyl terminal end, the technique of the present invention, which clones the complete gene, would not be subject to that problem. This could be exemplified by a cell-surface membrane antigen where the immune system only produced antibodies against amino terminal portions of the protein.
Finally, the technique of the present invention prepares DNA fragments containing the genes in direct proportion to the number of copies of that gene in the genome rather than to the relative amount of mRNA present in the biological sample available. This advantage overcomes the problem of cloning a gene for a protein which is present in only limited quantity or which is produced in only tiny amounts by a cell sample and which can be detected only by sensitive detection methods. This is so because the mRNA to make the cDNA for the gene of interest is normally present only in cells where the protein is made.
The other two commonly used techniques may not have the disadvantages of the cDNA technique, but in turn have disadvantages of their own. The random shear method does not suffer from the lack of mRNA, but like the cDNA technique most often results in the cloning of only a portion of the gene. Also due to the small sizes of the pieces of DNA used in this method a larger number of clones must be examined to find the gene.
The restriction endonuclease method of preparing DNA for cloning is usually a required second step for the cDNA and for random shear techniques to eventually clone the entire gene. It also may be used to clone genes directly, but has the disadvantage that numerous restriction enzymes must be tried to find just one (of .about.100) enzyme, which will cut the DNA in the right position to yield the gene or a portion of it in a form which the cloning vector can allow to be expressed. The reason for this is that a restriction enzyme inherently recognizes a specific 4 or 6 base pair sequence of the DNA and cuts the genomic DNA everywhere the recognition sequence is present. Since the DNA base sequence is stable, the position of these sites is fixed. For many genes it may be difficult or impossible to find the appropriate enzyme to cut the gene containing DNA fragment in a form allowing the gene product to be expressed.
The present invention provides advantages over the prior art techniques by first cutting the DNA at specific sites, less than 100 bases from the front and the rear of the coding area of the gene. It does not cut randomly within the gene. This results in DNA pieces containing the whole gene as opposed to fragments or small portions of a gene. The frequency of the appropriate DNA fragment in the clone mixture is dependent on gene copy number and the size of the DNA of the genome rather than the amount of mRNA available. Furthermore, the close proximity of the cut site to the start of the gene makes the DNA suitable for expression of the gene product in many expression vectors. Moreover, the cut is not specific for a certain base sequence, rather it recognizes a structure in the DNA and cuts somewhat randomly within a certain base stretch overcoming the problem present in many expression vectors of the DNA being in the right reading frame to allow the gene product to be expressed. The present invention is useful both for prokaryotic as well as eukaryotic genomes, hence is of general applicability.